TY - JOUR
T1 - Geminin inhibits a late step in the formation of human pre-replicative complexes
AU - Wu, Min
AU - Lu, Wenyan
AU - Santos, Ruth E.
AU - Frattini, Mark G.
AU - Kelly, Thomas J.
N1 - Publisher Copyright:
© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2014/10/31
Y1 - 2014/10/31
N2 - The initial step in initiation of eukaryotic DNA replication involves the assembly of pre-replicative complexes (pre-RCs) at origins of replication during the G1 phase of the cell cycle. In metazoans initiation is inhibited by the regulatory factor Geminin. We have purified the human pre-RC proteins, studied their interactions in vitro with each other and with origin DNA, and analyzed the effects of HsGeminin on formation of DNA-proteincomplexes. The formation of an initial complex containing the human origin recognition complex (HsORC), HsCdt1, HsCdc6, and origin DNA is cooperative, involving all possible binary interactions among the components. Maximal association of HsMCM2-7, a component of the replicative helicase, requires HsORC, HsCdc6, HsCdt1, and ATP, and is driven by interactions of HsCdt1 and HsCdc6 with multiple HsMCM2-7 subunits. Formation of stable complexes, resistant to high salt, requires ATP hydrolysis. In the absence of HsMCM proteins, HsGeminin inhibits the association of HsCdt1 with DNA or with HsORC-HsCdc6-DNA complexes. However, HsGeminindoesnot inhibit recruitment of HsMCM2-7toDNAto form complexes containing all of the pre-RC proteins. In fact, HsGeminin itself is a component of such complexes, and interacts directly with the HsMcm3 and HsMcm5 subunits of HsMCM2-7, as well as with HsCdt1. Although HsGeminin does not prevent the initial formation of DNA-protein complexes containing the pre-RC proteins, it strongly inhibits the formation of stable pre-RCs that are resistant to high salt.Wesuggest that bound HsGeminin prevents transition of thepre-RCto a state that iscompetentfor initiation of DNA replication.
AB - The initial step in initiation of eukaryotic DNA replication involves the assembly of pre-replicative complexes (pre-RCs) at origins of replication during the G1 phase of the cell cycle. In metazoans initiation is inhibited by the regulatory factor Geminin. We have purified the human pre-RC proteins, studied their interactions in vitro with each other and with origin DNA, and analyzed the effects of HsGeminin on formation of DNA-proteincomplexes. The formation of an initial complex containing the human origin recognition complex (HsORC), HsCdt1, HsCdc6, and origin DNA is cooperative, involving all possible binary interactions among the components. Maximal association of HsMCM2-7, a component of the replicative helicase, requires HsORC, HsCdc6, HsCdt1, and ATP, and is driven by interactions of HsCdt1 and HsCdc6 with multiple HsMCM2-7 subunits. Formation of stable complexes, resistant to high salt, requires ATP hydrolysis. In the absence of HsMCM proteins, HsGeminin inhibits the association of HsCdt1 with DNA or with HsORC-HsCdc6-DNA complexes. However, HsGeminindoesnot inhibit recruitment of HsMCM2-7toDNAto form complexes containing all of the pre-RC proteins. In fact, HsGeminin itself is a component of such complexes, and interacts directly with the HsMcm3 and HsMcm5 subunits of HsMCM2-7, as well as with HsCdt1. Although HsGeminin does not prevent the initial formation of DNA-protein complexes containing the pre-RC proteins, it strongly inhibits the formation of stable pre-RCs that are resistant to high salt.Wesuggest that bound HsGeminin prevents transition of thepre-RCto a state that iscompetentfor initiation of DNA replication.
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U2 - 10.1074/jbc.M114.552935
DO - 10.1074/jbc.M114.552935
M3 - Article
C2 - 25231993
AN - SCOPUS:84908701463
SN - 0021-9258
VL - 289
SP - 30810
EP - 30821
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 44
ER -